1. The present invention relates to 2-arachidonylglycerol (2-AG) to be used as inhibitor of a tumor necrosis factor (TNF-xcex1), in the reduction of edema caused by closed head injury, in the reduction of neurological deficits caused by closed head injury and stroke and in treating pathological conditions caused by TNF-xcex1 and/or by radical oxgen intermediates (ROI), in pharmaceutical composition for the same use comprising as active ingredient 2-AG, the use of 2-AG and pharmaceutical compositions comprising same in the preparation of a medicament for the treatment of said indications and methods of treatment by 2-AG and pharmaceutical compositions comprising same for diseases caused by said indications.
2. Two types of endogenous cannabinoids have been identified, the most thoroughly investigated compounds being arachidonylethanolamide (anandamide) and 2-arachidonylglycerol (2-AG) (Mechoulam and Ben-shabat, 1999). Although these endocannabinoids, in particular anandamide, have been the object of investigations in various systems, their physiological roles are not clear (Mechoulam et al., 1998). In view of the anti-inflammatory action of plant and synthetic cannabinoids, and of the presence of endocannabinoids and of cannabinoid receptors in organs associated with immune regulation, a plausible role attributed to the endocannabinoid system is an anti-inflammatory one. Indeed, anandamide has been shown to exhibit anti-inflammatory effects (Molina-Holgado et al., 1997) and 2-AG suppresses interleukin-2 through down regulation of the nuclear factor of activated T cells (Ouyang et al., 1998).
3. Tumor necrosis factor-xcex1 (TNF-xcex1) is involved in the pathogenesis of various immune mediated processes and is the key mediator in septic shock (Tracey and Cerami 1993). This cytokine is released mainly by mononuclear phagocytic cells in response to injection of lipoplysaccharide (LPS, an endotoxin derived from Gram negative bacteria) to experimental animals. TNF-xcex1 affects both the central nervous system and periphery. It causes fever, sickness behavior, anorexia, symphathetic discharge and stimulation of pituitary hormones. It can also induce programmed cell death in neurons.
4. Circulating TNF-xcex1 and other cytokines have been, shown to be transported into the brain and to affect its function. TNF-xcex1 can be synthesized in the brain in situ, mainly by microglia, but also by astrocytes, neurons and endothelial cells. TNF-xcex1 activity can be induced by ischemic and traumatic brain injury (Newton and Decicco, 1999 and ref. cited) and it plays a dual role in the pathophsiology of these injuries (shohami et al. 1999).
5. Since TNF-xcex1 is an important inflammatory mediator, inhibition of its production by a body constiuent may reflect a role for such an endogenous ligand in the anti-inflammatory processes of a living tissue. We now report that 2-AG inhibits in vitro TNF-xcex1 production both in-vitro, in murine macrophages, as well as in mice.
6. As cannabinoids have been shown to have antioxidative properties (Mechoulam et al., 1998) we also looked into the action of 2-AG on the formation of radical oxygen intermediates (ROI). These are highly toxic species that are formed in many biological reactions.
7. We investigated the effect of various concentrations of 2-AG (0.05-50 xcexcg/ml) on the production of TNF-xcex1 and nitric oxide generation by macrophages. These cells were chosen on the basis of their versatility, as they participate very actively in innate and immune functions. They phagocytize, ingest and destroy many infections agents, and act as effector cells in both humor and cell mediated immune responses. However, macrophages can also cause a wide array of inflammatory diseases.
8. Mouse peritoneal macrophages (harvested from C57BL/6 mice) were incubated in vitro with various concentrations of 2-AG, together with either LPS, which triggers TNF-xcex1 production, or with LPS and IFNxcex3, which together trigger nitric oxide generation. In the concentration studied (50 xcexcg/ml-0.5 xcexcg/ml) 2-AG was not toxic to macrophages assessed either by trypan blue or erythosin B dye exclusion. TNF-xcex1 production was determined by bioassay using BALB/c CL.7 cells as targets (Gallily et al., 1997).
9. ROI formation in macrophages was determined by a luminol-enhanced chemiluminescence response assay (employing biolumate LB 85, Belhold, Wildbad, Germany (Avron and Gallily, 1995).
10. For determination of the effect of 2-AG on TNF-xcex1 levels in serum of LPS-treated mice, female C57BL/6 mice 9-11 weeks old, weighing 20-23 gr were injected i.p. with either 5 mg/kg LPS alone or with LPS (5 mg/kg) and 2-AG (10 mg/kg) or with LPS (5 mg/kg) and 2-AG (1 mg/kg) 2-linoleoylglycerol (2-LinoG) (10 mg/kg) and 2-palmitoylglycerol (2-PalmG) (5 mg/kg). After 90 min they were bled and serum TNF-xcex1 activity (titer) was bioassayed, as described above.